Title: XII' Transcription
1(No Transcript)
2Transcription
A General Overview
- transcription in Prokaryotes and Eukaryotes are
similar, but a little more complicated /
sophisticated in eukaryotes - in bacteria, gene
control is carried out primarily by DNA binding
proteins that affect transcription - several
different types of transcriptional control
circuits exist Bacteria have no nucleus ? mRNA,
once generated is completely accessible to
ribosomes Since both TRANSCRIPTION TRANSLATION
occur 5 ? 3, bacterial protein synthesis can
proceed on an mRNA even as it is being synthesized
3Concurrent Synthesis ofmRNA Proteins in
Bacteria
RNA Pol.
3
mRNA
Ribosome
5
5
nascent protein
3 (C-terminus)
5 (N-terminus ATG start codon, Met)
4Overall Strategies in Regulating Gene Expression
in Prokaryotes
- since translation starts right away, initiation
of transcription is the critical point in gene
control (in eukaryotes, the mRNA is exported out
of the nucleus in a regulated manner, and
translation from it can be regulated) - the
concentration of a given mRNA (this dictates
protein production levels) species depends on two
factors a) The rate of synthesis b) The rate of
degradation. Typical life time of an RNA
molecule in prokaryotes is on the order of
minutes or less ? So once synthesis of an mRNA
stops, the mRNA quickly disappears resulting in a
stop in protein synthesis
5In general there are three elements of gene
control in prokaryotes
- Transcriptional initiation ? Most Important
- Transcriptional Termination ? Small effect
- Rapid RNA Turnover ? Important
- bacterial mRNAs are not chemically modified
before translation (eukaryotic mRNAs are spliced,
5-capped polyadenylated the major
modifications) - the lack of modifications allows
coupled transcription and translation to occur
kits are available for both eukaryotic and
prokaryotic coupled transcription-translation
6In eukaryotes, transcription occurs in the
nucleus, where no ribosomes are present
although ribosomal precursors mature in the
nucleolus - mRNAs also require covalent
modification before emerging into the
cytoplasm ? Coupled transcription and
translation cannot occur
Prokaryotes Eukaryotes also differ in the
apparent use of gene control Bacteria
gene-control allows a single cell to adjust to
its environment Eukaryotes (especially
metazoans) Some genes serve the above function
But another very important role for gene control
is regulation of a genetic program
Single cell ? multicellular organism with
differentiated tissues
7Prokaryotes - synthesis of RNA from a DNA
template. - first step in gene expression - needs
dsDNA template, RNA polymerase, NTPs Mg2
E. Coli RNA polymerase 5 subunits, ?2???
youre making RNA
Table 26.1
Core Enzyme (Elongation)
Holoenzyme (Initiation)
8Viral T7 RNA polymerase - single 98 kD subunit -
enzyme most commonly used in the lab to make RNA
- begins transcription from the so-called T7
promoter
- this promoter is present in many vectors to
allow for in vitro transcription
OR to allow specific expression of a gene in
bacteria harbouring T7 polymerase
9Some important definitions
coding or sense strand
1
template or anti-sense strand
- the coding strand of a gene has the same
sequence as the RNA produced BUT it is the other
(template) strand that RNA polymerase reads - RNA
polymerases (unlike DNA polymerases) do NOT
require a primer - The first nucleotide has a 5
triphosphate Usually RNAs start with a 5ppp-A or
5ppp-G
10Initiation
- Bacterial Polymerases - - transcription
begins at a promoter a specific DNA sequence -
RNA polymerase holoenzyme scans DNA (tracks along
DNA) - Binds promoter to give a closed
complex Footprinting reveal that RNA protects
nucleotides from 55 to 5 from nucleases. -
Melts 17 bp around -10 region ( open
complex) - RNA synthesis begins at 1 - At the
start of synthesis, ? is released.
11Initiation Elongation of Transcription
Closed Complex
- typically the first bases in a transcript is a
purine (A or G)
Open Complex
Initiation
Figure 26.6
12Promoter - the promoter consensus sequences
derived from averaging sequences from a large
number of bacterial promoters - actual sequence
at any promoter varies - poor matches to
consensus initiate transcription up to 300x less
frequently than at perfect matches - the ? factor
is needed only for initiation Without ?, core
enzyme can transcribe foreign DNA but theres
no specificity With ?, core is specific, only
initiates at promoters only transcribes
template strand
13S
mRNA
nucleotide nomenclature 1 first
transcribed nucleotide
1
2
3
-1
15-20
5-8
Figure 26.11 (Biochemistry)
14- variations in the promoter sequences have a
variety of effects on the promoters strength
Figure 26.12
15Elongation
- does not require ? factor - a Transcription
Bubble migrates down the DNA 17 bp of melted
DNA 12 bp of this paired with nascent RNA 1
turn of an A-type helix - no more than this to
prevent RNA from getting entwined in the DNA The
RNA-DNA mini-helix can rotate about the DNA
strand to constantly free the nascent
transcript - elongation moves at 50 bases/sec. -
No editing (unlike DNA synthesis) Accuracy 1
error in 105 bases Since mistakes are not passed
on to daughter cells, they are not critical
16The Transcription Bubble
Figure 26.8
17Termination
- Two Types
- Rho-Independent or Factor Independent
- Rho-Dependent
1) Rho-Independent Involves formation of a GC
rich hairpin in the nascent RNA strand, followed
by a run of Us The hairpin is thought to
promote RNA dissociation from the template The
poly U sequences leaves a weak residual
association ? RNA falls off
18Hairpin
Weak association AU base pairs are weaker than
GCs.
Figure 26.15
191) Rho-Dependent Rho is a bacterial
Termination Factor It acts as a hexamer of 46 kD
subunits - binds a specific 72 base sequence of
ssRNA It then hydrolyses ATP and eventually
disrupts pairing between the nascent strand
template
Figure 26.16